Isolation and usage of exosomes in central nervous system diseases

Abstract Background Exosomes are vesicles secreted by all types of mammalian cells. They are characterized by a double‐layered lipid membrane structure. They serve as carriers for a plethora of signal molecules, including DNA, RNA, proteins, and lipids. Their unique capability of effortlessly crossing the blood–brain barrier underscores their critical role in the progression of various neurological disorders. This includes, but is not limited to, diseases such as Alzheimer's, Parkinson's, and ischemic stroke. Establishing stable and mature methods for isolating exosomes is a prerequisite for the study of exosomes and their biomedical significance. The extraction technologies of exosomes include differential centrifugation, density gradient centrifugation, size exclusion chromatography, ultrafiltration, polymer coprecipitation, immunoaffinity capture, microfluidic, and so forth. Each extraction technology has its own advantages and disadvantages, and the extraction standards of exosomes have not been unified internationally. Aims This review aimed to showcase the recent advancements in exosome isolation techniques and thoroughly compare the advantages and disadvantages of different methods. Furthermore, the significant research progress made in using exosomes for diagnosing and treating central nervous system (CNS) diseases has been emphasized. Conclusion The varying isolation methods result in differences in the concentration, purity, and size of exosomes. The efficient separation of exosomes facilitates their widespread application, particularly in the diagnosis and treatment of CNS diseases.

0][11] Although the term "exosomes" is commonly used, it is recommended that the term "small extracellular vesicles (sEVs)" be substituted to accurately represent the diverse population of EVs. 12 Exosome biogenesis occurs in two distinct stages: first, membranous vesicles form through inward budding within endosomes, and then these vesicles are released into structures called multivesicular bodies. 13Exosomes can transport membranous and cytoplasmic components from their parent cells, including proteins, messenger RNA (mRNA), microRNA (miRNA), long noncoding RNA (LncRNA), lipids, metabolites, and even fragments of DNA. 14,15The contents and functions of exosomes from different cells are specific.
In the early days, people believed that the exosomes were the "scavengers" in the cells, responsible for carrying the wastes generated by cells. 16Afterward, the function of exosomes in the central nervous system (CNS) disorders attracted increasing attention with extensive research on the biological origin, material composition, transport mechanisms, intercellular signaling, and distribution in the body fluids of exosomes.Exosomes participate in intercellular communication 17,18 and regulate physiological and pathological processes. 19ey are used for diagnosing CNS disorders 20 and are also considered ideal candidates for drug delivery systems (DDSs) in the treatment of CNS diseases. 21Exosomes, as therapeutic agents or DDSs, offer the following advantages.Being naturally derived from cells, exosomes possess inherent biocompatibility and biodegradability.
This reduces the risk of immune rejection or adverse reactions, making them a safer drug delivery option compared with synthetic carriers. 22,23Moreover, their lipid bilayer structure not only provides stability but also shields the encapsulated cargo, ensuring efficient delivery to target sites. 24Exosomes can be engineered to carry specific targeting molecules on their surface.This feature enables exosomes to selectively deliver therapeutic cargo to the desired cells or tissues. 25ven the significance of exosomes in biomedical research, their isolation and purification are critical for comprehending their mechanisms of action and their potential applications in biomedical science.Exosome isolation methods refer to the techniques developed to enhance the concentration and quality of exosomes.Despite the recognized importance of exosomes, accurately characterizing their biological activity largely hinges on the efficiency of diverse exosome separation methods.Furthermore, the current outlook for clinical research on exosomes is not optimistic.The main reasons are as follows 1 : exosome mass production and purification have not yet been developed; and 2 clinical production of exosomes does not have an international standard.Hence, careful selection of an appropriate isolation method that allows for the efficient and reliable extraction of exosomes from diverse biomaterials and other EVs is essential.
Research on different exosome isolation technologies has become a hotspot in recent decades.These technologies can be broadly classified based on their underlying principles of separation, such as ultracentrifugation, size-based separation, polymer precipitation, immunoaffinity capture, and microfluidic separation, among others.
This study aimed to summarize the current research progress in exosome isolation through typical examples and the application of these isolation methods to the CNS.Subsequently, the characteristics, applications, advantages, and disadvantages of different methods were compared to assist researchers in selecting appropriate exosome isolation techniques.The results might provide a reference for improving the standardized process of exosome extraction and preparation, promoting the clinical transformation and application of exosomes.Finally, we have systematically summarized the application of exosomes in diagnosing and treating common CNS diseases, aims to provide a reference for future research on exosomes as diagnostic biomarkers and therapeutic carriers in CNS diseases.

| ISOL ATI ON OF THE E XOSOME S
The isolation and enrichment of exosomes from complex biological components play a vital role in both fundamental research and the advancement of clinical applications.However, the isolation and purification of exosomes from complex biological fluids are challenging due to their small nanoscale size and low buoyant density.Despite the obstacles encountered, researchers have made notable progress in exosome isolation using various techniques.These include ultracentrifugation, ultrafiltration, size exclusion, polymer-based precipitation, affinity capture, and microfluidic technology, among others (Figure 1A).And, numerous laboratories have successfully isolated exosomes using these diverse approaches. 26Nevertheless, the concentration, purity, and size of exosomes vary due to different isolation methods. 27,28This section describes exosome isolation based on various isolation principles, with a detailed description of each method (Figure 1B).The benefits and drawbacks of various isolation methods have been compared to aid researchers in choosing suitable techniques for exosome isolation based on their specific conditions and conducting relevant studies (Table 1).Good separation efficiency helps promote the widespread application of EVs, especially in the field of CNS diseases that require breakthroughs in the blood-brain barrier (BBB) (Figure 2).

| Exosome isolation based on ultracentrifugation
Ultracentrifugation is a classical method using powerful centrifugal force to isolate exosomes.Exosomes aggregate and settle at the bottom of the ultracentrifuge tube due to centrifugal force.This method is categorized into differential ultracentrifugation and density gradient ultracentrifugation, depending on the separation principle employed (Figure 1B-a).

| Differential ultracentrifugation
Differential ultracentrifugation is also known as ultracentrifugation.Ultracentrifugation is considered as the "gold standard" and one of the most commonly used and reported techniques for | 3 of 20 exosome extraction.The use rate of ultracentrifugation technology accounts for 56% of all exosome isolation technologies. 50e principle of ultracentrifugation is to isolate exosomes under different centrifugal forces according to the different densities and sizes of EVs in the bio-sample. 51The initial step in ultracentrifugation involves eliminating particles that possess a high buoyancy density, including cells, cell debris, apoptotic bodies, and biopolymer aggregates.This is achieved through a series of centrifugation steps: first, centrifuging at 300-400g for 10 min to settle the majority of cells; then, at 2000g to eliminate cell debris; and finally, at 10,000g to separate aggregates of biopolymers, apoptotic bodies, and other structures with a buoyant density greater than that of EVs. 52Following this procedure, the ultimate sedimentation of exosomes is carried out through centrifugation of samples at a speed of 100,000-200,000g for a duration of 2 h or a minimum of 70 min. 53tracentrifugation is a physical separation method with little effect on the biochemical components of exosomes.It is easy to use and suitable for isolating and extracting exosomes from largevolume samples; however, it is not appropriate for studying trace and valuable samples.In addition, the presence of contaminants, the long time required, the expensive equipment, and the large sample dose limit the effectiveness and use of ultracentrifugation in clinical trials and diagnostics.Exosomes can be damaged by excessively high rotational speeds, leading to a decrease in the activity of their contents such as RNA and DNA, used for subsequent analysis.

| Density gradient centrifugation method
Density gradient centrifugation is an improved method of traditional differential ultracentrifugation.It introduces an inert centrifugal medium in the centrifugal system, allowing exosomes to be transferred to the appropriate density gradient interval through ultracentrifugation.The medium of density gradient centrifugation is mainly sucrose and iodixanol.
The sucrose density gradient centrifugation method is to form a continuously distributed density gradient layer from low to high under the action of the centrifugal force.The exosomes are isolated by bands formed in different regions and concentrated in the density range of 1.13-1.19g/mL.cushion ultracentrifugation for isolating exosomes.They collected conditioned serum-free media from human mesenchymal stem cells (MSCs) cultured for 48 h to isolate the exosomes.They removed cellular debris by centrifugation at 300g for 10 min, followed by centrifugation at 10,000g for 30 min to eliminate microvesicles.Then, under the condition of using equal volumes of pre-processed conditioned media, exosome isolation was performed using both ultracentrifugation and one-step sucrose cushion ultracentrifugation methods.A relatively high recovery of exosomes with cup-shaped morphology was observed using the one-step sucrose cushion ultracentrifugation method, as determined by nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). 31 This approach effectively isolated exosomes from the denser vesicles, enabling the solubilization and removal of major impurities associated with urinary exosomes. 32When compared to the sucrose density gradient centrifugation method, the iodixanol density gradient centrifugation method offers several advantages, including lower viscosity, metabolic inertness, nontoxicity to cells, and better preservation of cell integrity and functionality.Li et al. introduced a robust method platform called cushioned-density gradient ultracentrifugation.They used a 60% iodixanol cushion for concentrating nanoparticles to enhance exosome recovery and maintain their physical integrity and biological activity.This cushion prevented pellet formation in the centrifuge tube.Additionally, exosome purification through density gradient ultracentrifugation effectively eliminated protein contaminants and non-exosome nanoparticles.
Moreover, iodixanol exhibited high biological inertness and compatibility with various downstream in vitro functional assays.It is also deemed suitable for animal studies, making it an excellent choice for reliable and efficient biochemical and physiological investigations of purified exosomes, eliminating the need for their removal. 54Konadu et al. devised a method to isolate exosomes from human immunodeficiency virus 1 (HIV-1) particles in the plasma of infected individuals using iodixanol velocity gradients.Despite their comparable size and density, exosomes were found to separate into the upper fractions of the iodixanol gradients, characterized by lower density, while viral particles were observed in the lower fractions with higher density. 33nsity gradient centrifugation enhances the purity of isolated exosomes using samples obtained via ultracentrifugation. 34wever, this method is complicated to operate, requires high technical proficiency from the operator, has low throughput, is time-consuming, and is not effective in removing lipoproteins and chylomicrons from blood samples.

| Size-based isolation method
The method of isolating exosomes based on their size has also been investigated.Ultrafiltration and size exclusion chromatography (SEC) isolate exosomes from other components in biological samples based on sizes.

| Ultrafiltration
Ultrafiltration is a technique that employs the pressure disparity across an ultrafiltration membrane to segregate exosomes according to their characteristic size.Ultrafiltration and membrane filtration are essentially synonymous (Figure 1B-b).Various ultrafiltration methods can effectively separate exosomes, including sequential filtration, tandem filtration, centrifugal ultrafiltration, and tangential flow filtration (TFF). 55These methods employ membrane filters with specific molecular weight or size exclusion limits, separating the suspended particles or polymers based on their size or molecular weight.
Syringe-based sequential and tandem filtration are deadend techniques.Sequential filtration involves multiple rounds of filtration, with each round using a different molecular weight cutoff.On the contrary, the tandem filtration method combines multiple filters within a single syringe.In this method, the exosomes, which typically have a size exclusion threshold ranging from 20 to 200 nm, are trapped within an intermediate membrane.
Centrifugal ultrafiltration, which integrates dead-end filtration and centrifugation, enables the isolation of exosomes using nanoscale pores.A nanoporous membrane is rotated within a tube using centrifugal force to facilitate the passage of sample content.
Before centrifugal ultrafiltration, centrifugation or dead-end filtration is commonly conducted at 0.22 μm to remove larger particles such as cells, cell debris, and protein aggregates, preventing blockages. 56Recently, TFF has emerged as a novel method for isolating exosomes.Unlike traditional approaches, TFF operates

| Size exclusion chromatography
The principle of SEC is based on different sizes.The sample flows through the column, and substances larger than the pore size of the gel particles cannot enter the pores.They are eluted through the space between the porous gel and the mobile phase.However, small molecules remain trapped in the gel pores and are eventually eluted by the mobile phase, requiring a longer elution time (Figure 1B-c).
The stationary phase or column can be packed with various gel polymers, including cross-linked dextran (Sephadex), agarose, polyacrylamide (Biogel P), or allyl dextran (Sephacryl). 53 date, the SEC method has been successfully used for the isolation, purification, and enrichment of exosomes from a wide range of biological fluids, for example, urine, 59 saliva, 60 serum, 61 and so on.The EVs in the supernatant of tumor cells consist of different types of vesicles, including exosomes, microvesicles, and apoptotic bodies.To distinguish tumor-derived exosomes (TEX) from non-TEX, researchers need to categorize the EVs into subgroups.This can be achieved by employing specific tumor cell culture conditions and using mini-SEC. 39,62Additionally, tissue exosomes, for example, from synovial tissue, can be extracted using SEC. 63SEC has become the preferred method for rapidly isolating relatively pure exosomes from plasma. 64Hong and colleagues successfully modified the SEC approach, which allows for simple and reproducible isolation from small plasma volumes (1 mL) of exosomes retaining the structural integrity and functional activity. 65,66majority of current reports on exosome isolation by size exclusion methods include a combination of SEC isolation with other isolation techniques.Yong et al. presented an approach that involved the preprocessing of plasma, combined with ultracentrifugation and SEC to isolate EVs and subsequently enrich exosomes, which enabled the use of a greater starting volume for exosome isolation, was highly reproducible and time-efficient, and provided a greater yield. 67The combination of ultrafiltration and SEC led to a significant enhancement in the exosome quantity and concentration compared with ultracentrifugation.Specifically, it resulted in a maximum 58-fold increase in the number of exosomes and a maximum 836-fold decrease in the concentration of co-purified soluble factors, taking into account the exosome yield. 68Yang et al. used Sepharose-4B to construct a custom SEC column for separating and characterizing exosomes.The self-made SEC column effectively segregated EVs from complex serum proteins, with EVs predominantly concentrated in fractions 8-13, exhibiting favorable morphology and yield.SEC proved to be superior in all aspects through a comprehensive comparison with the commonly employed ultracentrifugation and total exosome isolation commercial kit (Total Exosomes Isolation Reagent, TEI, 4478360, Invitrogen), striking a balance between isolation purity and yield. 69Nevertheless, the SEC method alone still possessed certain limitations and residual impurities.And when they used the SEC + ultracentrifugation approach ingeniously addressed the drawbacks of SEC and optimized the quality and purity of EVs derived from serum, surpassing the efficacy of either method used individually. 69Studies found that a more effective method could be achieved in terms of both efficiency and purity by implementing a modified serum sEVs isolation protocol, which involved two ultracentrifugation cycles and a 30% sucrose buffer. 34e advantages of SEC are its simple operation, good reproducibility, large sample size, and relatively uniform size of the isolated exosomes.The disadvantage is that it can isolate particles similar in size to others, resulting in reduced purity. 53Currently, commercial columns have been developed for isolating and purifying exosomes based on the principles of SEC. 70,71The commercial product qEV column of iZON based on the SEC principle can extract exosomes with high purity from 150 μL to 10 mL of initial samples within 15 min, which ensures the stability of biochemical components and morphological structure of exosomes while extracting efficiently (http:// www.moery bio.net/ Produ cts-34788 885.html).

| Separation technology based on polymer precipitation
More than half a century ago, the polymer precipitation method was used to enrich and purify viruses. 72This method was also used to extract exosomes due to the similar size and biochemical properties of exosomes and virus particles. 73The principle of polymer coprecipitation technology is that the hydrophilic polymer interacts with the hydrophilic bond of the exocrine body of the sample to form a hydrophobic microenvironment around the exocrine body so as to form a precipitate and extract exosomes (Figure 1B-d). 26The exosome precipitation isolation method isolates exosomes by introducing chemical reagents (mainly polyethylene glycol, lectin, and so forth) to change exosome dispersibility or water solubility.
Polyethylene glycol (PEG) is the most commonly used polymer for exosome isolation.It effectively promotes the enrichment and production of exosomes. 74Ludwig et al. developed and enhanced a precipitation method using PEG to concentrate EVs from cell culture supernatants.This method demonstrated that a significant amount of co-precipitated molecules such as bovine serum albumin could be effectively removed by washing the PEG pellet and reprecipitating it through ultracentrifugation. 75 The first stimuli-mediated isolation system for exosomes was developed by incorporating a thermalresponsive, reductant-cleavable copolymer onto the membranes of exosomes.This system used a thermal-responsive copolymer called poly N-isopropylacrylamide-co-N-acryloxysuccinimide (PNN), which was attached to the lipid bilayer of exosomes to modulate their surface wettability.When the temperature of the PNN-labeled exosome samples exceeded the lower critical solution temperature (LCST = 31°C), the attached PNN underwent a phase transition from hydrophilic to hydrophobic, leading to the spontaneous aggregation of PNN-labeled exosomes (PNN-Exos).Furthermore, the PNN conjugate could be detached from the exosomal surfaces through reductant-triggered S-S cleavage. 76Studies also found that when hydrochloride was introduced into milk to facilitate isoelectric precipitation, it effectively isolated and purified bovine milk exosomes.
This significant advancement contributed to the progress of research on the health management of dairy cattle and the development of DDSs in human medicine. 77ecipitation-based exosome isolation methods are most attractive for clinical research and most widely used for product formation on the market because of their simple operation and speed, no exosome damage, and low demand for additional equipment for separation.Over the past few years, the commercially available polymer-based precipitation technique has gained popularity due to its time-saving nature, potential for improved reproducibility, userfriendly approach, and ability to yield high-purity vesicles containing small RNAs.This method outperformed traditional centrifugationbased techniques, which varied in effectiveness depending on the user. 78,79Many commercial kits separate exosomes based on the principle of polymer precipitation.Although the precipitation method does not yield pure exosomes, which may contain substances that interfere with downstream experiments, it is still an effective way to achieve rapid enrichment and concentration of EVs in large-volume samples, saving both time and cost. 80With the continuous deepening of research, the polymer precipitation method has been continuously upgraded and improved, and the extraction effect is greatly improved.

| Separation technology based on immunoaffinity capture
A variety of specific proteins are found on the membrane of exosomes, such as the typical four tetraspanin proteins CD9, CD63, and CD81. 81This feature provides a new idea for separating and extracting exosomes: using antibodies or aptamers of exosomes on the membrane of the specific protein (antigen) to produce an immune response, facilitating the separation of exosomes.The principle of immunoaffinity technology is to separate and enrich exosomes by identifying specific proteins of exosomes (Figure 1B-e).Depending on the type of antibody substrate carrier, immune separation methods can be categorized into magnetic bead immune separation, chromatographic stationary phase separation, and other techniques.

| Magnetic bead immune separation
Magnetic particles, such as iron, nickel, neodymium, or magnetite, can be easily functionalized with biomolecules, such as antibodies.
This modification enables the magnetic particles to specifically attach to exosomes, facilitating their extraction from a complex matrix using magnetic actuation.This approach helps eliminate interference caused by the biofluid matrix, allowing for the preconcentration of exosomes and enhancing the sensitivity of the detection process. 44ion-exchange (AE) magnetic beads have been extensively studied for their potential in exosome isolation because of their rich ion-exchange capacity, high binding capacity, and fast magnetic response.Studies found that the AE-based exosome isolation method using these magnetic beads yielded highly efficient recovery rates (>90%) and resulted in exosome samples with high purities (14.42 × 10 10 particles/mg). 45 tion method, the SIMI system extracted approximately 59% more exosomes from the 293T cell culture medium, while also offering a shorter isolation time, higher purity, and improved biological activity. 82A novel method was developed to isolate exosomes from serum in a precise and time-dependent manner.This approach used magnetic beads for capturing exosomes and employed lightactivated elution.This was achieved by attaching a CD63 aptamer to the surface of the magnetic beads using a light-sensitive nitrobenzene group.The excitation of ultraviolet light was controlled at around 365 nm, and exosomes were selectively released from the beads while preserving their structural and functional integrity. 83e use of immunomagnetic beads for exosome separation has become popular due to their high specificity and convenient mag- effective release with an efficiency of up to 82.45%. 84Furthermore, the surface of TiO 2 contains a Lewis acid site that exhibits a robust affinity for phosphate ions, enabling it to selectively bind to intact biofilms.Consequently, TiO 2 is also extensively employed for the enrichment of biofilm-like substances.The use of TiO 2 microspheres for exosome separation demonstrates significantly superior efficiency compared with the conventional methods.This technique not only reduces sample processing time but also enhances separation specificity. 85A novel approach was introduced to isolate urinary exosomes by combining the selective interaction between exosomes and TiO 2 with ultrafiltration.This method effectively reduced nonspecific protein adsorption and expedited sample preparation.
Additionally, the isolated exosomes retained their structural integrity and could be easily lysed for subsequent analysis. 86

| Chromatographic stationary phase separation
Despite the availability of numerous exosome isolation options, alternative techniques that offer improved throughput and purity are strongly desired.Chemical separation and processing platforms, similar to those used in high performance liquid chromatography (HPLC), present a potential alternative approach.Hydrophobic interaction chromatography (HIC) can be employed to extract exosomes from seminal plasma obtained through intrauterine insemination (IUI) treatments. 46In 2019, Huang et al. developed a protocol for the isolation and quantification of human urinary exosomes using HIC on a polyester (PET) capillary-channeled polymer (C-CP) fiber stationary phase. 87ng and colleagues investigated the application of PET C-CP fibers in an HIC protocol for extracting exosomes from a human plasma sample.Their preliminary findings indicated that their method enabled the isolation of exosomes with similar yields and size distributions but at a significantly faster rate compared with the conventional isolation techniques.Moreover, it effectively reduced the presence of accompanying proteins and other impurities.Additionally, the C-CP HIC approach offered advantages over commercial exosome kits in terms of cost (at $5 per column) and reusability (approximately 30 times). 88A study conducted in 2021 employed C-CP fiber micropipette tips in an HIC solid-phase extraction process.This novel approach allowed quick (<15 min) and affordable (<$1 per tip) isolation at sample volumes and timeframes relevant to clinical applications. 89

| Others
In Immune capture separation has high specificity, high purity, and ease of use, while preserving the structure and morphology of exosomes.However, this method is limited to the isolation of exosomes with positive markers, resulting in low extraction efficiency.
In addition, the activity of exosomes can be easily affected by pH and salt concentration, making it unsuitable for large-scale exosome isolation.In addition, high cost and low production hinder its further development and use.

| Separation technology based on microfluidic control
Microfluidics  exosome capturing and analysis. 93The design enabled precise quantification of individual microbeads' fluorescence by capturing the average fluorescence density of each bead, effectively minimizing optical interference from background noise.Furthermore, a largescale microfluidic single-bead trapping device was used, facilitating exosome molecular analysis.This was achieved through on-chip elution and lysis of the purified sample, enabling downstream analysis.
Additionally, this design supported multiple surface modifications, allowing for the simultaneous capture and analysis of multiple exosomes, thus enabling multiplexed exosome capturing and analysis.
Over the past few years, a team of researchers developed and introduced a microfluidic system called exosome isolation and detection (EXID).In the isolation chamber, exosomes were isolated using immune magnetic beads and marked with anti-Programmed cell death 1 ligand 1(PD-L1) fluorescence probes.Subsequently, the exosome-labeled beads were guided into the analysis region and captured using micropillar arrays to measure the fluorescence of PD-L1.The EXID system successfully achieved integrated onchip processing, eliminating the need for off-chip operations.
Leveraging the micropillar array, the system enabled single-bead analysis, facilitating the detection of exosome heterogeneity.
This groundbreaking technology held immense potential as a tool for personalized cancer diagnosis and immunotherapy. 94 Furthermore, a comb-like structure made of nickel was devised to amplify the magnetic force exerted on immunomagnetic nanoparticles, resulting in a significant increase in the recovery rate of exosomes.Within the detection zone, the isolated exosomes were specifically labeled using fluorescent antibodies and subsequently detected using a confocal fluorescence microscope.As a proof-ofconcept demonstration for the ExoSD chip, exosomes with both high purity and high recovery rates were successfully isolated from cell culture supernatant at various flow rates (40, 60, 80, and 100 μL/min). 47Li et al. developed a novel method called the homogenous magneto-fluorescent exosome (hMFEX) nanosensor, which combined magnetic isolation and enhanced fluorescence measurement.This integrated approach enabled rapid and on-site analysis of TEXs.The hMFEX nanosensor exhibited high specificity and could detect TEXs across a dynamic range of five orders of magnitude.Additionally, it had a limit of detection of 6.56 × 10 4 particles/μL. 95spite the improved efficiency and purity achieved by labelbased exosome isolation methods, further advancements are needed to increase throughput and enable targeting of multiple EV populations with different types of labels.These techniques can be costly due to the expensive labeling procedures.Moreover, they are often complex and time-consuming due to the intricate steps involved in the labeling preparation and the need for washing steps to isolate the labeled exosomes.Furthermore, the capturing molecules used may introduce sample contamination and alter the properties of exosomes, potentially leading to inaccurate results in subsequent characterization processes and compromising the reliability of downstream analysis in clinical applications.

| Techniques for label-free isolation of exosomes
Size-based exosome isolation strategies are often studied because they offer unmarked separation, ensure size uniformity, and minimize sample bias.Chen and colleagues introduced a novel ultrafiltration approach that involved using dual coupled harmonic oscillations within a dual membrane filter setup to generate transverse waves.
This approach achieved clogless and ultrafast purification of exosomes, overcoming the limitations of other methods. 96Yang et al.
presented a novel microfluidic device capable of modulating the membrane pore size using ion-sputtered gold layers of varying thickness.This innovative approach enabled the effective isolation of exosomes from body fluids based on the pore size. 97Recently, Hua et al. designed a double TFF-based microfluidic device to separate and enrich exosomes.This device used porous membranes with pore sizes of 200 and 30 nm to sort vesicles ranging from 30 to 200 nm, enabling the isolation and purification of exosomes from conventional sources such as cell culture supernatants and human sera. 98ao et al. introduced an innovative approach automated centrifugal microfluidic disk system integrated with functionalized membranes (Exo-CMDS).This system aimed to isolate and enrich exosomes.
Subsequently, the enriched exosomes were subjected to a novel aptamer fluorescence system (Exo-AFS), to efficiently detect exosome surface proteins.Exo-CMDS yields highly qualified results, with an optimal exosomal concentration of 5.1 × 10 9 particles/mL from trace amounts of blood samples (<300 μL) in only 8 min through the centrifugal microfluidic disk system.This approach accomplished exosome isolation and purification in a single step, providing an accurate clinical diagnosis system for lung cancer. 48parating the exosomes from other components in the blood requires a significant amount of time and effort due to their small for size-based isolation of salivary exosomes.They found that the platform consistently isolated secretions from saliva samples for use in downstream saliva secretion liquid biopsy applications, regardless of viscosity changes and collection methods. 100Wu et al. presented a label-free and contact-free exosome isolation method based on acoustofluidics, which integrated acoustics and microfluidics to isolate exosomes directly from whole blood.The acoustofluidic platform consisted of two modules: a microscale cell-removal module designed to remove larger blood components and an exosome isolation module for subgroup separation of EVs.In the cell-removal module, 110-nm particles were successfully isolated from a mixture of micro-and nanosized particles, achieving a yield of more than 99%.In the exosome isolation module, a 98.4% purity of exosomes was achieved from the EVs mixture.The isolation of exosomes from whole blood was successfully achieved by seamlessly integrating the two acoustofluidic modules onto a compact chip, surpassing an impressive blood cell-removal rate of more than 99.999%.This device allowed for the rapid, biocompatible, label-free, contact-free, and continuous-flow isolation of exosomes.It provided a distinctive method to study the impact of exosomes on the development and advancement of human diseases.Moreover, this technology held promise for various applications such as health monitoring, medical diagnosis, targeted drug delivery, and personalized medicine. 101 summary, the conventional method for isolating exosomes requires a significant amount of sample, which can pose challenges when dealing with limited sample availability.Additionally, the iso-

| A COMPARISON OF E XOSOME ISOL ATI ON ME THODS AND THEIR APPLI C ATI ON IN ISOL ATING CN S E XOSO ME S
Various isolation methods are available for different purposes and applications.However, despite the development of numerous methods for exosome isolation and purification, some limitations still exist that cannot meet all requirements, which should be considered when planning experiments.The comparison between different isolation methods for exosomes is depicted in Table 1.Different isolation methods might introduce variations in the yield, purity, and integrity. 103Therefore, considering the advantages and disadvantages of these methods is crucial for downstream research applications. 104When selecting, combining, and optimizing methods, considering the starting material, equipment availability, therapeutic use, administration route, and desired end product is essential to achieve high yield and purity for clinical applications.Combining different isolation methods may yield better isolation results than using a single method.Studies have combined multiple methods for isolation and purification to enhance the yield and purity of exosomes, improving the isolation efficiency and enrichment. 105 have been reported, however, when isolating exosomes from typical samples of patients with CNS (e.g., 0.5 mL) for analysis, only a limited number of reported methods could generate sufficient exosomes.
In this study, we provided a summary and introduction of these successful methods used for isolating exosomes from samples of CNS origin (Table 2).Multiple centrifugation and ultracentrifugation steps could be used to isolate exosomes from the Glioblastoma multiforme (GBM) cell culture supernatant. 107In 2022, D'Acunzo et al. described a detailed protocol: an iodixanol-based high-resolution density gradient separation method.This technique allowed quantitative and reproducible analysis of brain EV subtypes in normal and pathological brain conditions, including neurodegenerative diseases such as AD and PD. 108Exosomes could also be isolated from the CSF of patients with amyotrophic lateral sclerosis and other neurological disorders using SEC. 109A well-established immunoprecipitation assay using anti-L1CAM-coated beads could be used to purify exosomes from the plasma of patients with PD and healthy controls. 110The systematic review and meta-analysis showed that the ExoQuick kit offered optimum isolation of exosomes for PD diagnosis. 111Table 2 shows that the isolation of CNS exosomes is currently mainly performed using more established commercial kit methods.Exosomes can also be isolated from brain tissue, which provide valuable and unique insights into the intricate workings of the brain.They offer data that cannot be obtained from exosomes isolated from conditioned media or body fluids. 131

| ADVAN CEMENTS IN E XOSOME RE S E ARCH FOR THE D IAG NOS IS AND TRE ATMENT OF CN S D IS E A S E S
In recent years, remarkable advancements have been made in studying exosomes and their role in intercellular communication.
Exosomes have emerged as key players in regulating network connectivity and transport within the CNS.In microglial exosomes, the level of miR-124-3p significantly increased from the acute phase to the chronic phase of traumatic brain injury (TBI).The upregulation of miR-124-3p targets a sub-family of the PDE4 family (PDE4B), inhibiting the activity of the Mammalian target of rapamycin (mTOR) signaling pathway, thereby suppressing neuronal inflammation and promoting neurite outgrowth. 132Exosomes derived from astrocytes have the potential to transport miR-17-5p, providing a protective shield for neonatal rats against hypoxic-ischemic brain damage (HIBD) by effectively inhibiting the expression of Homo sapiens BCL2 interacting protein 2 (BNIP2). 133In addition, specific components have been identified in exosomes, such as proteins, nucleic acids, and lipids, that can serve as indicators of disease progression or treatment response.By analyzing the composition and quantity of exosomes in biofluids such as CSF or blood, clinicians can gain valuable insights into the underlying pathology and monitor the effectiveness of therapies.Exosomes also have potential applications in CNS therapy.As a therapeutic agent or novel drug carrier, exosomes can cross the BBB and achieve effective drug delivery. 134,135Figure 2 shows the overview of exosomes in the diagnosis and treatment of CNS diseases.

| Application of exosomes in diagnosing neurological disorders
Using exosomes as potential biomarkers for CNS diseases is one of the main application areas.Exosomes have emerged as attractive targets for clinical diagnosis and biomarker discovery due to the following reasons.First, the contents of exosomes, including lipids, proteins, nucleic acids, and more, undergo changes during disease progression, potentially reflecting the disease status.Second, exosomes can be noninvasively isolated from easily accessible biological fluids such as blood, urine, and saliva.The noninvasive nature of exosome isolation is particularly important for early disease diagnosis, especially in the CNS.Third, exosomes possess a double-layered membrane that protects potential biomarkers from degradation.
Fourth, exosomes are highly stable, making them clinically feasible, as samples can be stored for extended periods before analysis.Fifth, exosomes carry markers associated with their cell of origin, allowing traceability.Finally, exosomes can cross the BBB, providing information about neural cells that would otherwise be difficult to obtain without invasive techniques. 136e blood is the only tissue in contact with all organs and carries a great deal of valuable information about the organism.Biomarkers derived from blood exosomes are highly sought after in the field of neurodegenerative diseases.Plasma exosomes were found to spread and cluster around β-amyloid plaques in an animal model of AD. 137 miR-193b, miR-135a, and miR-384 from plasma exosomes are aberrantly expressed in AD and may serve as potential biomarkers for diagnosing AD. 112 Serum exosomal miR-19b, miR-195, and miR-24 are aberrantly expressed in PD and may serve as potential biomarkers for PD diagnosis. 122Neuronal exosomes are used as a new source of biomarkers for neurodegenerative diseases.Neuronal-derived exosomal growth associated protein 43 (GAP43), neurogranin, synaptosome associated protein 25 (SNAP25), and synaptotagmin 1 serve as biomarkers that accurately reflect pathological changes in the AD brain and possess the ability to distinguish between AD and amnestic mild cognitive impairment (aMCI).Interestingly, when these biomarkers were combined, they could detect preclinical AD 5-7 years prior to the onset of cognitive impairment. 119The increased release of α-synuclein serum neuronal exosomes precedes the diagnosis of PD, persists as the disease progresses, and is associated with the aggregation of proteins, predicting and distinguishing PD from atypical Parkinson's syndrome. 123The aforementioned studies have demonstrated the potential of CNS-derived blood exosomes as a source of diagnostic, prognostic, and progression biomarkers for neurodegenerative diseases.In addition to blood products, exosomes from other biological fluids, such as CSF, saliva, and urine, have been used for diagnostic purposes.Exosomes have been detected in the CSF and cell culture media of humans as well as model species such as mice and monkeys. 138 proved to be a potentially cost-effective screening method for early disease detection. 139In addition, aberrant expression of calbindin and SNAP23 in urinary exosomes was also identified as a potential biomarker for the diagnosis of PD. 128 Table 2 summarizes the biomarkers used in exosomes from different sources for the diagnosis of CNS diseases and their corresponding methods of exosome isolation.
In summary, the aforementioned studies provided evidence for the potential of exosomes as biomarkers for CNS diseases; using exosomes as molecular diagnostic markers had promising applications.However, at the same time, some issues still need to be considered.The first consideration is the reliability of exosomes as CNS biomarkers.Only small amounts of pathogenic proteins cross the BBB and move to the periphery, and the levels of pathogenic proteins in the blood are influenced by many organs.Therefore, the levels of pathogenic proteins in plasma neuron-derived exosomes may not be sensitive to small pathological biological changes in the brain. 140The second consideration is the reliability and consistency of the experimental results.The principal steps of various exosome extraction methods are inconsistent, the exosome quantification methods applied are not uniform, and these problems may lead to poor reproducibility of the experiments.In conclusion, the research on exosomal biomarkers is still in its early stages, and it is hoped that these issues will be addressed in future studies, thus providing new support for the diagnosis.

| Application of exosomes in treating neurological disorders
The transportation of large protein molecules is limited due to the restrictive nature of the BBB, resulting in the inability to use many drugs that can effectively treat various neurological disorders in clinical settings.However, exosomes are vesicles derived from cells and enclosed by a double-layered lipid membrane, demonstrating the remarkable ability to traverse the BBB effectively. 141Exosomes possess the capacity to deliver their cargo directly to recipient cells.
This feature makes them well-suited as therapeutic agents, including engineered exosomes, which offer advantages that soluble factors alone cannot achieve.Moreover, exosome-based treatments exhibit enhanced safety compared with cell-based therapies involving therapeutic cell transplantation and offer regulatory advantages, further supporting their potential as a viable treatment option for neurological disorders.

| Cell-free therapy based on stem cell-derived exosomes
Exosomes themselves can be used as a therapeutic approach for CNS diseases. 142Exosomes derived from MSCs (MSC-EVs) can cross the BBB at the tissue level and promote fusion with the cell membrane, allowing the direct release of their contents into the cytoplasm.This mechanism effectively avoids capture by endosomes/lysosomes and enables the exosomes to traverse organelle membrane barriers. 143Cs are better producers of EVs than other kinds of cells and possess various biological functions. 144The potential therapeutic approaches of MSC-EVs for neurodegenerative diseases are as follows: enhanced neurofunctional recovery, 145 remyelination, 146 synaptic plasticity, 147 and neuroprotection. 148In addition, injections of MSCderived EVs have been reported to enhance the resistance of hippocampal neurons to the detrimental effects of oxidative stress and β-amyloid (Aβ)-induced synaptic damage. 149These functions play a multifaceted role in regulating the pathophysiological processes of  All drug-loading techniques have their advantages and disadvantages. 157Using advanced engineering techniques to engineer exosomes has improved the efficacy of exosomes as agents for disease treatment.
In the field of AD treatment, engineered exosomes have been shown to have a pivotal role.AD is clinically characterized by progressive dementia caused by the pathological accumulation of neurofibrillary tangles and Aβ plaques.Notably, tau hyperphosphorylation and Aβ hyperaggregation have been identified as two crucial features in the AD brain, even occurring decades before the onset of cognitive dysfunction. 17Studies showed that beta-site APP-cleaving enzyme 1 (BACE1) was a protease responsible for the N-terminal cleavage of the amyloid precursor protein, which generated the βamyloid peptide.When exosomes were designed to express neurontargeting peptides on their surface and injected into the blood of mice after loading siRNA by electroporation, they exhibited a significant ability to cross the BBB, resulting in a significant reduction in the BACE1 expression.nerve growth factor (NGF). 163 Exosome-loaded resveratrol formulations for treating multiple sclerosis, which effectively suppressed inflammation in both the CNS and peripheral system, led to significant improvements in the clinical progression of diseases. 164In zebrafish, the incubation method enabled the loading of exosomes with doxorubicin and paclitaxel, allowing them to cross the BBB for brain cancer treatment.When administered separately, both drugs showed no absorption by the brain. 165In addition, mice treated with and (III) enhancing modified strategies of MSC-EVs to improve their biological functions and target site accumulation. 168Exosomes as DDSs offer a range of advantages: biocompatibility, biodegradability, intercellular communication capabilities, targeted delivery, immunomodulatory properties, low immunogenicity, low toxicity, and potential for personalized medicine.Among these, the potential for personalized medicine is an exciting aspect of exosomes.They can be isolated from specific cell types, enabling tailored cargo and targeting strategies that align with individual patient needs.This personalized approach holds great promise for advancing precision medicine.
These attributes position exosomes as a promising avenue for innovative therapeutic interventions.However, currently no completed or published clinical trials have reported on the role of exosomes in treating CNS diseases; but several clinical trials and studies that use exosomes as biomarkers are ongoing, which are worth noting.
For instance, using miRNA-manipulated microglial exosomes shows potential as a novel therapy for TBI and other neurologic diseases.
Specifically, miR-124-3p holds promise as a therapeutic target for mitigating neuronal inflammation following TBI. 132Exosomes as DDSs targeted miR-210 through exosomes for angiogenic therapy following cerebral ischemia in mice. 169Evox Therapeutics, under the leadership of Tony De Fougerolles, established strategic collaborations in 2020 with industry leaders such as Takeda and Eli Lilly.
These partnerships focused on harnessing the potential of exosomebased therapies to address unmet medical needs in the fields of rare diseases and neurological disorders, respectively. 170 summary, the isolation of exosome has yet to find a widely

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing not applicable to this article as no datasets were generated or analysed during the current study.

F
I G U R E 1 The classification of isolation methods of exosomes and the schematic diagram of their isolation principle.(A) The classification of isolation methods of exosomes.(B) The schematic diagram of exosomes isolation method principle.
Recently, Guo et al. developed a Streptag II-based immunomagnetic isolation (SIMI) system.This system used modified capture antibodies on magnetic nanoparticles, enabling specific and reversible recognition between Strep-Tactin and Strep-tag II.Compared with the gold standard ultracentrifuga- addition to the methods of immunomagnetic bead separation and chromatographic solid-phase separation based on immunoadsorption for exosome isolation, other methods based on the principle of immunoadsorption for exosome isolation have been reported.Lee et al. devised a cost-effective and user-friendly point-of-care testing platform, known as paper-based enzyme-linked immunosorbent assay (p-ELISA), to target EVs/exosomes.They achieved this by employing the streptavidin agarose resin-based immobilization (SARBI)technique.This innovative SARBI p-ELISA held great potential for application in resource-constrained regions/countries.90Barati et al.   devised a novel coaxial nanofiber configuration to achieve highly effective exosome isolation from bodily fluids.This configuration comprised a polycaprolactone polymer core and a sub-10-nm gelatin shell.The gelatin shell exhibited remarkable sensitivity to temperature fluctuations, enabling the efficient release of captured exosomes at the physiological temperature of 37°C.Moreover, the thin gelatin layer served to minimize contamination of the isolated exosomes.The electrospun nanofibrous membrane's interconnected micro-pores offered a substantial surface area for immobilizing specific antibodies, thereby facilitating the efficient capture of exosomes.91 is a signal detection-based exosome extraction technology.Currently, microfluidic methods for exosome separation can be broadly categorized into two main techniques: affinity-based (label-based) and label-free isolation methods (Figure 1B-f).

2. 5 . 1 || 9 of 20 WANG
Techniques for the label-based isolation of exosomesLabeling-based techniques for exosome isolation, usually by immunocapture, isolate exosomes from the bio-sample.Hisey et al. presented a novel herringbone-grooved microfluidic device chemically modified with antibodies targeting general and cancer exosome membrane biomarkers, specifically CD9 and EpCAM (epithelial cellspecific marker).This innovative device enabled the efficient isolation of exosomes from small quantities of high-grade serous ovarian cancer serum.92Tayebi et al. developed an immunoaffinity-based et al.method for exosome purification by specifically targeting a major protein marker (CD63) on exosomes, and this innovative method efficiently isolated and allowed visualization of exosomes enriched on the surface of microspheres.The design allowed accurate fluorescence quantification of individual microbeads by capturing the average fluorescence density of each bead, thereby effectively reducing optical interference from background noise.Additionally, using a large-scale microfluidic single-bead trapping device facilitated molecular analysis of exosomes through on-chip elution and lysis of the purified sample, enabling downstream analysis.Further, this design allowed multiple surface modifications, enabling the simultaneous capture and analysis of multiple exosomes, facilitating multiplexed Yu et al. proposed a highly integrated exosome separation and detection (ExoSD) chip, which represented a significant advancement building upon previous research.This chip offered a rapid and efficient solution for isolating and detecting exosomes.The microfilter structure within the separation zone underwent optimization to achieve a high level of purity in exosome isolation.
volume and low buoyancy density.Alternating current electrokinetic (ACE) microarray chip devices have a significant potential to address the challenges of exosome isolation and subsequent analysis of associated RNA, DNA, and protein biomarkers.The DEP force from the ACE microarray separates nanoparticles from fluid based on their dielectric properties.Ibsen et al. demonstrated the ACE microarray chip's ability to rapidly isolate and recover glioblastoma exosomes from undiluted human plasma samples.This device functions with a minimal plasma sample (30-50 μL) and can concentrate the exosomes around the ACE microelectrodes within 15 min.A simple buffer wash removes most plasma materials, leaving the exosomes concentrated on the microelectrodes.The entire isolation process, including on-chip fluorescence analysis, is only completed in under 30 min.99Exosome separation efficiency has also been improved by combining two or more isolation strategies.Acoustic waves are known for their high precision and biocompatibility in manipulating cells and other bioparticles.However, current acoustic-based isolation strategies can only process pre-treated biological fluids.This limitation necessitates additional equipment and time, increasing the risk of sample loss.Furthermore, current acoustic separation strategies can only distinguish between two types of targets, making it challenging to isolate exosomes directly from complex multicomponent fluids, such as undiluted blood.Wang et al. developed an acoustofluidic platform (the fusion of acoustics and microfluidics) lation and detection of samples are processed independently, causing potential inefficiencies.Numerous procedures are involved, from the initial isolation and purification of exosomes to the final identification via morphology and molecular biology.Therefore, implementing an efficient and straightforward integrated method for ExoSD is crucial.By monitoring exosomes in real time, microfluidics can quickly isolate exosomes and diagnose early noninvasive diseases, demonstrating promising application prospects in improving recovery, reducing sample volume, and shortening treatment time.102Label-free isolation methods for exosomes have several advantages, including lower cost, simpler and faster processing, and greater reproducibility.Additionally, unlabeled isolation can reduce potential sample contamination and allow the separated exosomes to maintain their natural characteristics, which does not affect downstream analysis and better supports the development of exosome-based applications.
During the 2013 International Society for EVs conference, scholars suggested combining ultrafiltration technology with SEC to enhance the extraction purity of exosomes. 106The pathogenesis of CNS diseases is multifaceted and can be categorized based on their underlying mechanisms.Neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease, are characterized by progressive degeneration of neurons.On the contrary, autoimmune diseases, such as multiple sclerosis and immune-mediated encephalitis, involve an abnormal immune response targeting the CNS.A reliable and reproducible method is necessary for isolating small EVs from the brain serum, cerebrospinal fluid (CSF), and other biofluid samples to comprehensively explore the physiological and pathological roles of exosomes in the brain.Various methods for isolating exosomes CSF exosomal RNA molecules are reliable biomarkers with fair robustness regarding specificity and sensitivity in differentiating PD from healthy and diseased (AD) controls.Aberrant expression of miR-153, miR-409-3p, miR-10a-5, let-7g-3p, miR-1, and TA B L E 2 Exosome isolation methods and their corresponding exosomal markers for the diagnosis of CNS diseases.kit (Invitrogen),112 ExoQuick (System Biosciences),113 Exosome Isolation Q3 kit for serum (Wayen Biotechnologies)114 SNAP-25, miR-193b, and miR-30b-5p N Y Human Total Exosome Isolation kit (Invitrogen), 112 Exosome Isolation Q3 kit for serum (Wayen Biotechnologies), 114 ExoQuick (System Biosciences) 115 miR-34b, miR-125b, miR-130b, miR-22-3p, miR-378a-3p, miR-135a, and miR-384 Synuclein, miR-195, miR-24, let-7d, miR-15b, miR-24, miR-142-3p, miR-181c, and miR-, miR-409-3p, miR-10a-5, and let-7 g-3p -3p in CSF-derived exosomes has emerged as a potential biomarker for the diagnosis of PD. 126 Collecting saliva from patients is less invasive than collecting blood, and saliva is easier to handle because it does not clot.However, saliva has been studied much less than plasma or serum as a source of biomarkers in neurodegenerative (and systemic) diseases, probably because of the higher variability and risk of contamination in saliva compared with blood.Rani et al. observed significant differences in the concentration of salivary exosomes among groups of patients with cognitive impairment and AD (P = 0.0023) compared with a healthy control cohort.They further demonstrated a novel method based on TEM technique to directly correlate the concentration of salivary exosomes with the progression of cognitive impairment in AD.This innovative approach CNS diseases, making MSC-EVs a promising therapeutic strategy for improving neurological function recovery.Bone marrow MSC-derived exosomes (BMSC-exos) administered through intracerebroventricular injection into a mouse model of AD induced by streptozotocin demonstrated that BMSC-exos could improve AD-like behavioral manifestations in mice.This improvement may be associated with the modulation of glial cell activation by BMSC-exos and the neuroinflammatory and hippocampal BDNF-related neuropathological changes. 150MSC-derived exosomes have been successfully used as a potential therapy to combat the progression of PD and improve symptoms, resulting in similar therapeutic effects as MSC transplantation. 151Li et al. discovered that exosomes derived from MSCs carrying miR-188-3p inhibited the expression of inflammasomes and autophagosomes by targeting the 3' Untranslated Regions (UTR) of Nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing 3 (NLRP3) and cyclin-dependent kinase 5, thereby alleviating nigral damage in a 1-methyl-4-phenyl-1,2,3,6-tetrahy dropyridine-induced PD mouse model. 152Chen et al. demonstrated that the exosomes secreted by human umbilical cord MSCs reached the substantia nigra via the BBB, alleviated apomorphine-induced asymmetric rotation, reduced nigral dopaminergic neuron loss and apoptosis, and upregulated dopamine levels in the striatum, which could treat PD.

Exo-cur or
exosomes loaded with signal transducer and activator of transcription 3 (Stat3) inhibitor JSI124 (Exo-JSI124) via intranasal administration were protected against lipopolysaccharide-induced encephalitis and experimental autoimmune encephalomyelitis induced by myelin oligodendrocyte glycoprotein peptide and exhibited delayed brain tumor growth in the Glioma 26 (GL26) tumor model.166 5 | DISCUSS IONExosomes, as a widely existing bioactive substance involved in intercellular information transmission, play a vital role in physiological and pathological processes.As an emerging field of research in recent years, the classification, generation mechanism, and characteristics of exosomes have also been gradually recognized.However, the separation and identification of exosomes is difficult due to their small size and low density, which is an important factor restricting research and transformation.Establishing a mature, stable, convenient, and fast method for the separation of exosomes is a premise for their use in medical diagnosis, prediction, and the pharmaceutical field.Many kinds of exosome isolation techniques have been developed.Each separation method is based on a unique property of exosomes, such as size, density, compressibility, surface protein, and so forth.However, these separation techniques still have problems such as low recovery, low purity, and possible contamination, seriously affecting the analysis of downstream results.The selection of methods for extracting exosomes from body fluids should be based on the characteristics of body fluid types, sample volume, follow-up research needs, and laboratory conditions.At the same time, rather than relying on a single method for extraction, using a combination of different methods to leverage complementary advantages is currently a wiser choice.Moreover, the study of exosomes in CNS diseases is an exciting and rapidly evolving field.Exosomes, derived from host cells, CSF, blood, and so on, contain DNA, RNA, proteins, and other molecules, providing insights into host cell attributes.Consequently, exosomes are promising indicators for the early detection of neurological ailments.Scientists can obtain information aiding in the early identification and detection of neurological disorders by analyzing exosome contents.Additionally, MSC-EVs show promise as personalized medicines for neurological disorders.However, their efficiency, homogeneity, and stability need to be ensured before they can be established as effective therapeutic tools.Not all EVs derived from MSCs are equivalent. 167Several strategies are necessary to enhance the potential therapeutic effects of MSC-EVs, such as,: (I) developing platforms for large-scale, cost-effective production of MSC-EVs to meet clinical needs; (II) improving detection methods for MSC-EVs to determine the optimal dosage, frequency, and tissue distribution; recognized and efficient method.The isolation technology and subsequent identification methods of exosomes still need continuous improvement.Exosome research and application has two main technical challenges: simplifying extraction and increasing production, and developing effective methods to distinguish exosomes from other EVs, especially functional vesicles.This study comprehensively analyzed the current research progress in exosome isolation strategies.It thoroughly introduced the existing technologies for exosome isolation and offered insights into the development of new methods for efficiently isolating exosomes from various bio-sample.Exosomes hold great potential for improving diagnosis, monitoring disease progression, and developing innovative therapeutic strategies, with promising applications in clinical diagnostics.However, some issues still need to be resolved in future research.More advanced separation techniques are needed for exosome identification and mass production.The mechanisms by which exosomes regulate the CNS are not fully understood.Moreover, surface modification and drug loading of exosomes remain a challenge.ACK N OWLED G M ENTS This study was supported by the National Natural Science Foundation of China (No. 82272347) and the Capital Health Research and Development of Special Fund (No. 2022-1G-2161).
Comparing various techniques for exosome isolation.
Gupta et al. compared differential ultracentrifugation and a modified method called one-step sucrose TA B L E 1

E 2
The overview of exosomes in the diagnosis and treatment of CNS diseases.
156Exosomes derived from dental pulp stem cells alleviated neuroinflammation in middle cerebral artery occlusion (MCAO) mice by inhibiting the high-mobility group box 1/toll-like receptor 4/ myeloid differentiation factor 88/nuclear factor-κB signaling pathway, thereby reducing ischemic brain injury.154Guo et oundthat BMSC-exos alleviated apoptosis of hippocampal neurons in rats and improved symptoms in a rat model of depression by upregulating the expression of miR-26a.155Moreover,exosomesfrom human BMSCs may be used as a safe noninvasive treatment to ameliorate behavioral symptoms of patients with autism spectrum disorders.156 160Moreover, targeting the phosphoryla-The findings revealed that Exo-Que demonstrated superior efficacy compared with free quercetin in alleviating the symptoms of AD.160PD is the second most common neurodegenerative disorder of CNS after AD. Theself-assembled nano carrier PR-EXO/PP@Cur, developed by Peng et al., enables intranasal delivery of curcumin loaded with exosomes overcame the limitations of MSC-EVs in 159Similarly, quercetin(Que) has shown potential in preventing tau pathology and providing neuroprotection, leading to improvements in cognitive and functional symptoms associated with AD.However, the clinical application of Que has been hindered by its limited brain targeting and low bioavailability.Qi et al. developed exosomes loaded with quercetin (Exo-Que) to enhance the drug's bioavailability and its targeting to the brain.ing, and delivered mRNA into the cytoplasm of target cells, facilitating efficient intercellular communication without the need for exosome concentration.Furthermore, the EXOtic device had potential utility in RNA-based therapeutic applications; designer exosomes could alleviate neurotoxicity and neuroinflammation in both in vitro and in vivo PD models. 162Except for AD and PD, engineered exosomes also have better therapeutic effects for other CNS diseases.Engineered exosomes loaded with neurotrophic factors and modified with rabies virus glycoprotein (RVG) ligand peptides could selectively target the damaged cerebral cortex in ischemic stroke, alleviating post-stroke inflammation and promoting neuronal survival through the action of . Sanchez JI, Jiao J, Kwan SY, et al.Lipidomic profiles of plasma exosomes identify candidate biomarkers for early detection of hepatocellular carcinoma in patients with cirrhosis.Cancer Prev Res (Phila).2021;14(10):955-962. 2. Cheng J, Nonaka T, Wong DTW.Salivary exosomes as nanocarriers for cancer biomarker delivery.Materials (Basel).2019;12(4):654.3. De Maio A. Human urine exosomes: another important member of the liquid biopsy family.Methods Enzymol.2020;645:195-208.4. Zhang Y, Xie Y, Hao Z, et al.Correction to "umbilical mesenchy- 1mal stem cell-derived exosome-encapsulated hydrogels accelerate bone repair by enhancing angiogenesis".ACS Appl Mater Interfaces.2022;14(12):14834-14835.